Applications of human pre-B-cell leukemia homeobox 1 (PBX1) gene and related drugs thereof
A gene and drug technology, applied in the use of human PBX1 gene and its related drug fields, can solve the problem that the role of PBX1 gene is not elucidated.
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Embodiment 1
[0067] Example 1: Preparation of RNAi lentivirus against human PBX1 gene
[0068] 1. Screening for effective siRNA targets against the human PBX1 gene
[0069] The PBX1 (NM_002585) gene information was retrieved from Genbank; an effective siRNA target for the PBX1 gene was designed using the design software Genechem of Shanghai Jikai Gene Chemical Technology Co., Ltd. In the coding sequence (CDS) region of the PBX1 gene, a sequence of 21 bases is obtained starting from every other base, and Table 1 lists 29 effective siRNA target sequences for the PBX1 gene.
[0070] Table 1 is targeted at the siRNA target sequence of human PBX1 gene
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[0073] Aiming at the siRNA target (taking SEQ ID NO: 27 as an example), synthesize a double-stranded DNA Oligo sequence (Table 2) containing AgeI and EcoRI restriction sites at both ends; act on pGCSIL- GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., figure 1 ), to linearize it, and identify ...
Embodiment 2
[0091] Embodiment 2: Real-time fluorescent quantitative RT-PCR method detects the silencing efficiency of PBX1 gene
[0092] Human lung cancer H1299 cells, liver cancer SMMC-7721 cells, breast cancer MCF-7 cells, human gastric cancer SGC7901 cells and human pancreatic cancer Panc-1 cells in logarithmic growth phase were digested with trypsin to make cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (the MOI of SGC7901 and H1299 is 10, the MOI of Panc-1, SMMC-7721 and MCF-7 is 20) value, add the virus that the suitable amount embodiment 1 prepares, change culture medium after cultivating 24h, wait for After the infection time reached 5 days, the cells were harvested. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV instruction manual of Promega Company, RNA was reverse-transcr...
Embodiment 3
[0100] Example 3: Detection of proliferation ability of tumor cells infected with PBX1-shRNA lentivirus
[0101] Human lung cancer H1299 cells, liver cancer SMMC-7721 cells, breast cancer MCF-7 cells, human gastric cancer SGC7901 cells and human pancreatic cancer Panc-1 cells in logarithmic growth phase were digested with trypsin to make cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, MOI of H1299 and SGC7901 is 10, MOI of Panc-1, SMMC-7721 and MCF-7 is 20), add appropriate amount of PBX1-shRNA virus, change medium after culturing for 24 hours, and wait for infection After the staining time reached 5 days, the cells of each experimental group in the logarithmic growth phase were collected. The complete medium was resuspended into a cell suspension (2×10 4 / ml), inoculate a 96-well plate at a cell density of about 2000 / well. ...
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